Craniofrontonasal syndrome in a male due to chromosomal mosaicism involving EFNB1: further insights into a genetic paradox.

Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by inactivating mutations in the gene for ephrin-B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/- females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ∼42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells overexpress ephrinB1 cluster and sort out from wild-type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.

Abnormal sterol metabolism in holoprosencephaly: studies in cultured lymphoblasts.

BACKGROUND: Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans. The aetiology is heterogeneous and remains unexplained in approximately 75% of patients. OBJECTIVE: To examine cholesterol biosynthesis in lymphoblastoid cell lines of 228 patients with HPE, since perturbations of cholesterol homeostasis are an important model system to study HPE pathogenesis in animals. METHODS: An in vitro loading test that clearly identifies abnormal increase of C27 sterols in lymphoblast-derived cells was developed using [2-(14)C] acetate as substrate. RESULTS: 22 (9.6%) HPE cell lines had abnormal sterol pattern in the in vitro loading test. In one previously reported patient, Smith-Lemli-Opitz syndrome was diagnosed, whereas others also had clearly reduced cholesterol biosynthesis of uncertain cause. The mean (SD) cholesterol levels were 57% (15.3%) and 82% (4.7%) of total sterols in these cell lines and controls, respectively. The pattern of accumulating sterols was different from known defects of cholesterol biosynthesis. In six patients with abnormal lymphoblast cholesterol metabolism, additional mutations in genes known to be associated with HPE or chromosomal abnormalities were observed. CONCLUSIONS: Impaired cholesterol biosynthesis may be a contributing factor in the cause of HPE and should be considered in the evaluation of causes of HPE, even if mutations in HPE-associated genes have already been found.

WHO Expert Committee on Biological Standardization.

This report presents the recommendations of a WHO Expert Committee commissioned to coordinate activities leading to the adoption of international requirements for the production and control of vaccines and other biologicals and the establishment of international biological reference materials. The report starts with a discussion of general issues brought to the Committee's attention and provides information on the status and development of reference materials for various antibodies, antigens, blood products and related substances, cytokines, growth factors, and endocrinological substances. The second part of the report, of particular relevance to manufacturers and national regulatory authorities, contains recommendations for the production and quality control of meningococcal group C conjugate vaccines, guidelines for regulatory expectations for clinical evaluation of vaccines, guidelines for the production and quality control of inactivated oral cholera vaccines and guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products.

Securing viral safety for plasma derivatives.

The various mechanisms that contribute to the viral safety of therapeutics prepared from human blood plasma are examined. The basic principles behind the 2 most important mechanisms for viral safety, inactivation, and removal are discussed, together with the main issues linked to manufacturing. Validation of virus safety and protein integrity is considered. Currently available methods for inactivation and removal of viruses are reviewed, followed by an overview of new and experimental methods that may have some promise for the future. This review ends with a brief discussion of the impact fractionation pool size may have on product safety and the potential new threat presented by contamination with prions.

Very long-chain acyl-CoA synthetases. Human "bubblegum" represents a new family of proteins capable of activating very long-chain fatty acids.

Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids. Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities. The most recently identified family is the very long-chain acyl-CoA synthetases (VLCS). Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins. It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases. Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity. Northern blot analysis showed that hsBG is expressed primarily in brain. Compared with vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates. Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation. Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others. Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound. Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active. hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acyl-CoA synthetases revealed that hsBG along with the D. melanogaster and murine homologs comprise a new family of acyl-CoA synthetases. Thus, two protein families are now known that contain enzymes capable of activating VLCFA. Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis.

New developments in plasma fractionation and virus inactivation.

Ethanol fractionation has been used for fifty years to prepare therapeutic proteins from human plasma. Although the method has been very successful, there are now newer techniques available, which might at least in part replace ethanol fractionation. Whether or not this will occur depends on the economic benefits the new methods offer. Although transmission of viruses through blood products is nowadays fortunately a rare event, the search for alternative inactivation and/or removal methods for viruses continues. The small, non-enveloped viruses are particularly difficult to deal with, and any new method should address this problem with a high priority. Transmission of spongiform encephalopathies (Creutzfeld-Jakob and similar diseases) by blood and blood products has not been observed so far. Preliminary experimental evidence indicates that the putative causative agent(s) are removed from the final products during fractionation.

Virus inactivation by pepsin treatment at pH 4 of IgG solutions: factors affecting the rate of virus inactivation.

BACKGROUND: IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material. STUDY DESIGN AND METHODS: The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content. RESULTS: Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. In contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus. CONCLUSION: Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.

Effects of intravenous infusion of lipid-free apo A-I in humans.

Apolipoprotein (apo) A-I is the principal protein component of the plasma high density lipoproteins (HDLs). Tissue culture studies have suggested that lipid-free apo A-I may, by recruiting phospholipids (PLs) and unesterified cholesterol from cell membranes, initiate reverse cholesterol transport and provide a nidus for the formation, via lipid-poor, pre-beta-migrating HDLs, of spheroidal alpha-migrating HDLs. Apo A-I has also been shown to inhibit hepatic lipase (HL) and lipoprotein lipase (LPL) in vitro. To further study its functions and fate in vivo, we gave lipid-free apo A-I intravenously on a total of 32 occasions to six men with low HDL cholesterol (30 to 38 mg/dL) by bolus injection (25 mg/kg) and/or by infusion over 5 hours (1.25, 2.5, 5.0, and 10.0 mg.kg-1.h-1). The procedure was well tolerated: there were no clinical, biochemical, or hematologic changes, and there was no evidence of allergic, immunologic, or acute-phase responses. The 5-hour infusions increased plasma total apo A-I concentration in a dose-related manner by 10 to 50 mg/dL after which it decreased, with a half-life of 15 to 54 hours. Coinfusion of Intralipid reduced the clearance rate. The apparent volume of distribution exceeded the known extracellular space in humans, suggesting extensive first-pass clearance by one or more organs. No apo A-I appeared in the urine. Increases in apo A-I mass were confined to the pre-beta region on crossed immunoelectrophoresis of plasma and to HDL-size particles on size exclusion chromatography. Increases were recorded in HDL PL, but not in HDL unesterified or esterified cholesterol. Increases also occurred in LDL PL and in very low density lipoprotein cholesterol, triglycerides, and PL but not in plasma total apo B concentration. These results can all be explained by combined inhibition of HL and LPL activities. Owing to the effects that this would have had on HDL metabolism, no conclusions can be drawn from these data about the role of lipid-free apo A-I in the removal of PL and cholesterol from peripheral tissues in humans. The kinetic data suggest that the fractional catabolic rate of lipid-free apo A-I exceeds that of spheroidal HDLs and is reduced in the presence of surplus PL.

Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

Internalization and degradation of monoclonal antibody chCE7 by human neuroblastoma cells.

Internalization and cellular degradation of a chimeric monoclonal anti-neuroblastoma antibody (MAb chCE7) is described. Immunofluorescence localization showed temperature-dependent redistribution of MAb chCE7 from the cell surface at 0 degrees C to vesicular structures after heating to 37 degrees C. SKN-AS cells which were pulse-labelled at 0 degrees C with radioiodinated chCE7 released 50% of the initial cell-bound radioactivity into the medium after 20 hr at 37 degrees C. Low-molecular-weight radioactivity in the medium was identified as iodotyrosine and the major intracellular degradation product was found to be an antibody fragment of 43 kDa. Degradation was blocked by chloroquine, an inhibitor of lysosomal function. MAb chCE7 binds to a carbohydrate epitope, as treatment with tunicamycin abolishes cell binding. In adherent cells in culture, inhibition of protein synthesis by cycloheximide affected cell-surface binding sites for chCE7 in a biphasic manner, with an initial increase followed by long-term decrease. Expression of binding sites was also found to be inhibited by sodium azide, by the lysosomotropic drug chloroquine and by Brefeldin A, a drug which prevents export of newly synthetized proteins to the cell surface. When cells were treated with high doses of chCE7 up to 24 hr and cell-surface binding sites were then measured after an acidic buffer wash, no loss of surface binding sites was found, indicating that pretreatment with MAb chCE7 does not induce down-regulation of its binding sites.

No evidence for protein modifications in fresh frozen plasma after photochemical treatment: an analysis by high-resolution two-dimensional electrophoresis.

To study if photochemical treatment of human plasma by methylene blue in combination with visible light induces protein alterations, we analysed by high-resolution two-dimensional gel electrophoresis (2-D PAGE) untreated and photochemically treated fresh frozen plasma collected by apheresis from the same donor (n = 8). Polypeptides were separated according to their charges by isoelectric focusing and to their sizes in polyacrylamide gels in presence of sodium and to their sizes in polyacrylamide gels in presence of sodium dodecyl sulphate, and visualized by silver staining. Despite their apparent complexity, protein maps of untreated plasma and photochemically treated fresh frozen plasma revealed identical spot patterns. Thus, photochemical treatment did not induce apparent protein pattern modifications.

Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters.

To further define the role of fibrin(ogen) and fibronectin in Staphylococcus aureus adherence to central venous catheters, the amount, chemical integrity, and biologic activity of these proteins adsorbed on lines inserted in hospitalized patients were prospectively studied. Polyurethane cannulas promoted a significantly lower adherence of S. aureus than polyvinyl chloride (P < .01) or Hickman (P < .001) cannulas and contained the lowest amount of immunologically assayed fibronectin but not of fibrin(ogen). Fibrinogen showed an extensive loss of adherence-promoting activity on inserted cannulas, which was related to its proteolytic breakdown, as detected by SDS-PAGE and immunoblots with antifibrinogen antibodies and confirmed by in vitro studies with purified protein fragments. In contrast, either intact or fragmented fibronectin, although present in much lower amounts than fibrin(ogen), could actively promote S. aureus adherence onto intravenous catheters.

Partitioning and inactivation of viruses during isolation of albumin and immunoglobulins by cold ethanol fractionation.

Albumin solutions invariably transmitted infectious hepatitis viruses before the introduction of pasteurisation in the final container. Immunoglobulin solutions (the older intramuscular as well as the current intravenous ones), on the other hand, only rarely transmitted hepatitis. The apparent safety of the latter was usually attributed to the presence of neutralizing antibodies and to the fractionation process. It was shown that viruses tend to concentrate in those fractions of the cold ethanol precipitation procedure which are used neither for albumin nor for immunoglobulin preparations. Additionally, ethanol alone inactivates some viruses, albeit much less at low temperatures than at room temperature. According to EC-directives, all manufacturers of stable blood products must introduce production steps which inactivate viruses or they have to prove that certain production steps, which are already being used, do inactivate viruses. In either case, the inactivation has to be validated with appropriate experiments. Procedures that are now recognized as virucidal are, e.g., pasteurisation (i.e., heating of the liquid product at 60 degrees C for 10 hours), solvent/detergent (S/D) treatment, photodynamic treatment, or incubation at pH4 with pepsin.

Production and characterization of a mouse/human chimeric antibody directed against human neuroblastoma.

Hybridoma CE7 produces a murine antibody (gamma 1/kappa) which binds to a 190-kDa cell-surface glycoprotein of human neuroblastoma. Because of its tumor specificity, it has been used routinely in clinical pathology to confirm diagnosis of neuroblastoma. We have isolated the gene segments coding for the variable regions of the immunoglobulin H and L chain of this hybridoma. These V genes were used to construct mouse/human chimeric H and L chain genes (gamma 1/kappa) which were then expressed in SP2/0 cells. A cell-binding inhibition assay showed that the specificity of the chimeric CE7 antibody (chCE7) is identical to that of the original CE7. Radioiodinated chCE7 binds to approximately 43,000 sites per neuroblastoma cell with an affinity of 10(10) M-1. In neuroblastoma-bearing nude mice, biodistribution studies with [125I]chCE7 were performed and tumor accumulations of up to 32% of injected dose/g tissue together with low blood and organ uptake were found.

Radioimmunolocalization of neuroblastoma xenografts with chimeric antibody chCE7.

This study was performed to evaluate the tumor targeting ability of chCE7 with a view to clinical applications in neuroblastoma imaging and therapy. A chimeric (mouse/human) monoclonal antibody (chCE7) of gamma 1/kappa isotype directed against a neuroblastoma-associated cell-surface glycoprotein is described. In vitro chCE7 binds with high affinity (KD approximately 1 x 10(-10) M) to SKN-AS human neuroblastoma cells. Binding studies with 125I-labeled chCE7 show temperature-dependent modulation of antigen binding and indicate that a proportion of the bound antibody is internalized due to rapid antigen turnover. In vivo biodistribution of radioiodinated chCE7 in nude mice bearing SKN-AS tumors shows optimal tumor uptake after 24 hr with about 30% of the injected dose per g. Optimal tumor/blood ratios (3.4:1) are reached after 4-5 days. Uptake in other organs including the reticuloendothelial system is low with tumor/organ ratios of 10 and more. Tumor uptake of chCE7 and the parent murine CE7 are found to be similar. Stability of chCE7 during and after radiolabeling is good with no loss of immunoreactivity in preparations labeled with 123I up to 100 mCi/mg and 80% immunoreactivity after labeling with 13 mCi/mg of 131I. Neuroblastoma xenografts can be imaged by radioimmunoscintigraphy with 123I- and and 131I-labeled chCE7.

Alterations in cell surface carbohydrate composition of a human colon carcinoma cell line affect adhesion to extracellular matrix components.

The adhesion of HT29 human colon adenocarcinoma cells to different extracellular matrix components was studied. While treatment of the cells with sialidase had no detectable effect on binding to laminin and fibronectin, attachment to collagen IV was decreased. However, additional removal of beta-(1-4)-bound galactose led to significantly reduced binding to all of the substrates, including fibronectin and laminin. Tunicamycin treatment, monitored by lectin-induced aggregation, drastically diminished cell adhesion to laminin and fibronectin, whereas cell binding to collagen IV was not affected. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion to collagen IV. The results show that a serine-containing RGD-related peptide GRGDSP has virtually no effect on colon carcinoma cell adhesion to type IV collagen. In contrast, when serine was substituted for threonine (GRGDTP) adhesion to collagen IV was strongly inhibited. After incubation of sialidase-treated cells with the threonine-containing peptide adhesion was almost totally blocked. These results demonstrate the existence of both RGD-dependent and carbohydrate-based mechanisms for metastatic human HT29 cell binding to collagen IV.

Virus inactivation during production of intravenous immunoglobulin.

The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families--human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)--were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.

Sialic acid dependent cell adhesion to collagen IV correlates with in vivo tumorigenicity of the human colon carcinoma sublines HCT116, HCT116a and HCT116b.

Cell surface sialylation of three metastasizing sublines HCT116, HCT116a and HCT116b of a human colon carcinoma was shown to correlate with their in vivo tumorigenicity. Lectin binding studies revealed further differences in cell surface glycosylation between HCT116a and HCT116 sublines. Binding to collagen IV correlated with the in vivo aggressiveness of the cells, whereas binding to fibronectin did not. On a laminin substrate the most tumorigenic line adhered best, but binding of the other lines was similar. Sialidase treatment of the cells had no effect on cell binding to laminin and fibronectin, but resulted in a decrease of cell binding to collagen IV.